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Essential role of MD-2 in TLR4-dependent signaling during Helicobacter pylori- associated gastritis

MD-2 在幽门螺杆菌相关性胃炎期间 TLR4 依赖性信号传导中的重要作用

基本信息

批准号：
15590647
负责人：
ISHIHARA Shunji
金额：
$ 2.24万
依托单位：
Shimane University (2004)Shimane Medical University (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Toll-like receptor 4(TLR4), a member of pattern recognition receptors, is the main receptor of lipopolysaccharide(LPS). MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. H.pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated here the role of MD-2 in TLR4-dependent signaling in H.pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H.pylori infection and 4 gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-κB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H.pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-κB and IL-8 promoter upon stimulation with H.pylori LPS. Live H.pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H.pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H.pylori LPS in the stomach.

Toll样受体4（TLR 4）是脂多糖（LPS）的主要受体，属于模式识别受体。MD-2与细胞表面上的TLR 4物理结合并赋予LPS应答性。幽门螺杆菌内毒素是引起胃炎的主要毒力因子之一。我们在此证实了MD-2在幽门螺杆菌相关性胃炎中TLR 4依赖性信号传导中的作用。本研究使用了从有和没有幽门螺杆菌感染的患者和4种胃癌细胞系中收集的胃活检样品。采用RT-PCR方法检测TLR-4和MD-2在组织学标本和细胞系中的表达。TLR-4在组织切片中的定位通过免疫组织化学进行评价。对于体外功能测定，我们建立了表达TLR 4和MD-2的AGS细胞的稳定转染子。流式细胞术检测TLR 4在细胞内的分布。报告基因分析法检测NF-κB活化和IL-8、MD-2启动子的活化。H.pylori感染可上调胃黏膜TLR 4和MD-2的表达。TLR 4染色主要在上皮细胞中观察到，位于细胞质和顶端表面。MD-2转染AGS细胞后，H.pylori LPS刺激细胞表面TLR 4表达增加，NF-κB和IL-8启动子激活增强。活幽门螺杆菌也刺激MD-2的转录激活。本研究揭示，在幽门螺杆菌感染期间，胃上皮细胞中MD-2表达升高，表明TLR 4/MD-2系统是参与胃中对幽门螺杆菌LPS的应答的有效受体复合物。